Challenging Gram Stains

Challenging Gram Stains
Linda Zuchowski, BS, MT(ASCP)SM
Microbiology Manager, Quest Diagnostics, Inc.
Denver, CO
Lenexa, KS
St. Louis, MO
ASCLS-MO/CLMA
Conference,
St. Louis, MO
March, 2016
No disclosures
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Objectives
 Recognize challenges that impact quality of Gram
stains (GS).
 Identify GS quality processes to overcome challenges,
and maximize positive patient outcomes.
 Review case studies utilizing telemicroscopy to
support or change diagnosis and treatment options for
infectious disease.
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Role of Gram Stain
 Integral tool in microbiology and infectious disease.
 Infectious disease among top 3 causes of death in US.
 Hospital acquired infections:100,000 deaths, $6 billion.
 CA-MRSA: 89,000 cases annually, $8 billion.
 Antibiotic resistance is among top 5 public health care
concerns: 2 million illnesses, 23,000 deaths annually.
 CDC plan: Target pathogen, treat infection, not
contamination or colonization.
 GS can help target pathogen and treatment!
Brinsley K et al. A J Infect Control 2005;33(1) 53-54.
Tom Frieden, Director CDC 2014. www.cdc.gov/mrsa/statistics/index/html accessed 4/14/14.
Antibiotic Resistance Threats in the US 2013, Executive Summary, CDC HHS.
www.nih.gov.
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Assess
specimen
quality
Diagnostic
&
therapeutic
value
Healthcare
benefits
Gram
stain
Positive
patient
outcome
Cost
effective
Challenging Gram Stains
Culture
correlation
Predictive
value
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Quality is Not Optional
Anything less than accurate, clinically
relevant results “is below the
community standard of care.”
Baron, Ellen Jo, Miller, J Michael, et al. IDSA Guidelines, A Guide to
Utilization of the Micro Lab for Diagnosis of Infectious Diseases:
2013 Recommendations by the Infectious Diseases Society of
America and ASM. CID 2013:57 August 15.
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Inadequate Diagnostics
“…More often, physicians must use incomplete or
imperfect information to diagnose an infection and thus
prescribe an antimicrobial just-in-case, or prescribe a
broad-spectrum antimicrobial when a specific antibiotic
might be better.
These situations contribute to selective pressure and
accelerate antimicrobial resistance.”
2008 www.NIH.gov
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Health Care Challenges Impact GS
 Affordable health care, financial restrictions.
 Micro lab consolidation trend – core lab.
 Satellite lab generalists responsible for critical Gram
stains: CSF, blood, sterile fluids, tissues.
 Less expertise
 Lower GS accuracy and correlation rate
 Less communication with off-site providers, labs
 Standard of care required.
Sautter Robert, Thomson Richard Jr, Consolidated Clinical Micro Labs, JCM, May 2015 vol. 53 no.5 1467-1472.
Ansara MK, Experience and recommendations for consolidating a micro lab Clin Microbiol Newsl. 2002;24(3):17-23
Barenfanger, Joan, et al., Interp of GS for the Nonmicrobiologist, LabMed, July 2001, No 7, Vol 32..
Susan Sharp, Elder, et al., Comp Assessment in the Clin Micro Lab, Clin Micro Reviews, July 2004.
Church, Deirdre et al, Quantitative GS Interpretation Criteria Used by Micro Labs in Alberta, Canada, JCM Nov 2000,
vol 38, no 11, 4266-4268.
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Gram Stains at Multiple Sites
Hospital
A
Clinic E
Stat Lab
D
Core
Micro
Lab
Hospital
B
Hospital
C
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Goal: Overcome GS Challenges
 Provide accurate, clinically relevant results
 Attain CDC goal of targeting pathogen, therapy
 Meet community standard of care
 Meet CAP requirements for GS
 Maximize positive patient outcomes
 Save health care costs.
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Implement GS Quality Plan
 Measure accuracy rate for baseline, set goal
 Improve GS quality and expertise.
 Partner with core lab
 Enhance training and competency program
 Increase frequency of competency exercises
 Review slides with major discrepancies
 Provide feedback
 Consider telemicroscopy.
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1st Scenario
55 year old female
patient with flank pain,
community hospital ER.
Dx: r/o kidney stone.
Previous UTI – Proteus,
on antibiotic therapy.
Positive blood culture
Gram stain = ??
Telemicroscopy review
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Antibiotic Effect
 Sub-inhibitory concentrations of antibiotics can produce




abnormal, pleomorphic bacteria morphology.
Penicillin can cause:
 Gnrs to elongate and produce filaments
 Pneumococci to form rods or globules
 Staphylococci to enlarge and become irregular
 Gonococci to form globules
Body fluid antibiotic levels can cause effect.
Gram stains challenging.
Collect specimens prior to therapy.
Lorian et al., “Abnormal Forms of Bacteria Produced by Antibiotics”, AJCP, Vol 64, Nov 1975.
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2nd Scenario
 Elbow abscess Gram stain = rare WBC, no
organisms seen.
-
Aerobic and anaerobic culture final = No growth.
 Physician questioned negative results.
 Patient Chart review:
 Day 1 - Pt admitted, elbow infection, Clindamycin initiated.
 Day 2 - Levofloxacin added.
 Day 3 - Ertapenem and Vanc started.
 Day 4 - Elbow abscess drained for C&S.
3rd Scenario
Pleural fluid smear too thick,
no organisms seen.
Nocardia grew on culture.
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Quality Specimen + Quality GS +
Competent Tech = Accurate Results
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Stain Quality
Evaluate specimen.
Avoid contamination, sterilize slide.
Cytospin fluids.
Make 2 smears.
Avoid Gram-variability:
-Methanol fixation – no heat!
- Counterstain longer for anaerobes
-Reagent concentration
Iodine (non-stabilized)
Decolorizer
Garcia L et al., Clin Micro Proc Handbook, Chap 3, ASM.
Chapin-Robertson et al., Cytospin Increases Sensitivity… of
CSF, JCM 30:377-380, 1992.
Zuchowski, L., GramStainology™: Gaining Proficiency in
Diagnostic Interp and Results Reporting, ASCP WLP 2015.
Challenging Gram Stains
Illustration by Kelly Zubeck.
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Gram Stain Quality – Wow!
Heat fixed
Methanol fixed
Zuchowski, L., GramStainology™: Gaining Proficiency in Diagnostic Interpretation and Results
Reporting, ASCP WLP 2015.
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Stain Quality Issue
Overdecolorized cocci?
Gram-variable Bacillus?
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Errors in Interpretation: Human Error
and Cell Wall Changes
 57 of 8,253 positive blood culture gram stains
were misread in 2 yr period = 0.7%.
 0.1% were Gram neg organisms staining
gram positive (Acinetobacter).
 1.3% were Gram pos organisms staining
gram neg (Bacillus and Clostridium).
 Gram variable staining due to cell wall
changes with loss of viability.
Rand, Kenneth et al., Errors in Interpretation of Gram Stains from Blood Cultures, AJCP,
2006;126:686-690. Dept of Path and Lab Med, Div of ID, Univ of FL,Gainesville
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Errors in Interpretation of Gram Stains
from Blood Cultures
12% Acinetobacter stain
Gram positive
AJCP
8% Bacillus species stain
Gram negative
AJCP
Rand, Kenneth et al., Errors in Interpretation of Gram Stains from Blood
Cultures, AJCP, 2006;126:686-690. Dept of Path and Lab Med, Div of Infectious
Disease, Univ of FL, Gainesville.
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Challenging Gram Stains
Clinically Relevant Reporting
 “No information is better than misinformation.”*
 Describing organism genus is more useful than
just morphology description.
 Avoid vague GS results: GPC, GNB
 GS should guide culture work-up.
*Raymond Bartlett, MD., Medical Microbiology: Quality Cost and Clinical Relevance, 1974
Baron E. et al., IDSA and ASM Guidelines 2013.
Bartlett R et al, Interp and Reporting of Organisms in Direct Smears, 1982. JAMA 247:857-59.
21
Challenging
Stains14:195-201
Bartlett R.,et al. 1991. Diagnostic Micro
InfectGram
Disease
Predictive Value of Staph or Strep
 Staph – 98% sensitivity and 100% specificity for
GPC in grapelike clusters.
 Strep – 100% sensitivity, 98% specificity for GPC in
pairs and chains
 Strep pneumo- 75% sensitivity and 97% specificity.
 Gram stain gave presumptive diagnosis for 80% of good
quality specimens.
 Why report just “GPC”?
Roson, B, et al., Clin Infect Disease 31:869-74, 2000
Aggar, Maki, et al., Efficacy of direct Gram stain in differentiating Staph and Strep in
Challenging
Gram Stains
blood cultures positive for GPC.
JCM 1978.
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Describe“GPC” Suggesting
Staph, Strep or Strep pneumo
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Describe“GPB” Suggesting
Clost/Bacillus, Branching, Diphth
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Predictive Value of GNB
 Differentiation of GNB reliable:
 Hemophilus–10% prevalence in symptomatic patients


Sensitivity 76%, Specificity 95-100% for GNCB.
PPV 100%, NPV 96%
 Enterics – 82% for blunt GNB.
 Pseudomonas – 56% for slender, sausage-link GNB.
 Why report just GNB? Target the pathogen.
Sadeghi, E., Matlow, A., et al., Utility of GS of sputa in cystic fibrosis , JCM p 54-58, Vol 32, No 1, Jan 1994.
Bartlett R et al, Interp and Reporting of Organisms in Direct Smears, 1982. JAMA 247:857-59.
Bartlett R.,et al. 1991. Diagnostic Micro Infect Disease 14:195-201.
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Describe “GNB”: Suggesting
Enterics, Fuso, Hemo
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Encapsulated GNB Challenge
Pantoea (Enterobacter) agglomerans
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GNDC compared to short GNB
Neisseria
Acinetobacter
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Short gnr - Acinetobacter
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CDC Public Image Library
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Enteric GNB – short
E coli
Acinetobacter
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GS Consistency Challenges:
 Variability
 Non-standard specimen
 Smear, stain quality
 Subjectivity
 GS interpretation.
 CAP MIC.11350
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Automated Stainers: One Solution for
Consistent Stain Quality
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CAP MIC.11350 GS Consistency
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CAP MIC.21530 GS Correlation
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Culture Correlation - Accuracy
 Depends on GS quality and expertise.
 Never 100%, but up to 97% for proficient techs.
 99.3% for blood cult GS read by experienced techs.
 57 of 8,253 blood cult GS misread in 2 years=0.7%*
 50% sputum cultures clinically misleading without GS
correlation.
 Appropriate monotherapy 94% of time when guided by GS.
Strand, CL., Positive Blood Cultures, Can We Always Trust the Gram Stain? Am J Clin Path 2006,
126:671-672.
*Rand, Kenneth H, et al., Errors in Interpretation of Gram Stains from Positive Blood Cultures, AM J
Clin Pathol 2006, 126:686-690.
Reed, W., Byrd, G., Gates R., et al, A Meta-Analysis of Sputum GS ,West J Med 1996; 165:197-204.
McCarter, Yvette, PhD, D(ABMM), ASM 2011 Clinical Core Curriculum III, Best Practices in the Work
Up of Resp Cultures.
1
Challenging Gram Stains
GS Correlation with Wound Culture
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GS Correlation with Sputum Culture
GS Correlation with Tickbite Culture
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4th Scenario
NICU blood culture GS
reported as budding yeast Blood Culture= viridans Strep
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GS Correlation QA
 Include in lab QA policy. (CAP MIC.21530)
 Monitor extreme discrepancies:
 Negative GS, but positive culture
 Positive GS, negative culture
 Bench tech must correlate results.
 Follow up, GS review, feedback.
 Having Micro tech review previously read slides is best
indicator of the tech’s GS interpretation proficiency.*
 Consider telemicroscopy…
*Munson, E, Block T., et al, Mechanism to Assess GS Interp Prof of Techs at Satellite Labs, JCM Nov 2007, vol 45, no 11; 3754-3758.
Dallas, Steven, Do Your GS Match Your Growth, Practical Response to CAP MIC.21530, ASM Clin Micro Portal Feb Hot Topic, 2014.
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Local QA Plan: Improve GS
Proficiency in Satellite Lab with
Telemicroscopy
 Review each GS daily or in real time with core
micro lab.
 Evaluate slide/stain quality and interpretation.
 Track correlation – accuracy rate.
 Monitor revised reports – provide feedback.
 Share expertise.
 Maximize GS results!
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Telemicroscopy Success
 Improved accuracy to >97% for 1000 slides!
 Results maintained since 2011.
 Increased confidence among non-micro techs.
 Rare revised reports!
 Win-win!
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Telemicroscopy
 There is growing interest for rapid, remote, expert
consultation.*
 Easy and cost effective.
 Allows real-time slide review with experts 24/7.
 Builds confidence for non-micro techs, beginners.
 Improves competency, accuracy, correlation.
 Evidence based, increased interpretive reporting.
 Email or print images, create image library.
*Rhoads, D., Sintchenko, V., Rauch, C., Clinical Microbiology Informatics, Clin. Microbiol. Rev.
October 2014 vol. 27 no. 4 1025-1047.
McLaughlin WJ, Schifman RB, Ryan KJ, et al., Telemicrobiology: feasibility study. Telemed J 1998
Spring;4(1):11-7. Accessed 3-22-15 www.mcbi.nlm.nih.gov/pubmed/9599069.
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Telemicroscopy Advantages
 Enhance collaboration with health care partners.
 Public health consultation – share parasite images
www.cdc.gov/DPDx (e.g.Cyclospora outbreak 2013).
 Bioterrorism preparedness (alternative to STATPack™)
 Boost QA program to meet CAP standards:
 Allows satellite labs to keep blood cultures on-site.
 Utilize in any dept with microscopy.
 Contributes to positive patient outcome.
 Benefits entire health care system.
Campbell, Sheldon et al, The Clin MicroChallenging
Lab in Diag
of LRT Infections, JCM Vol 49, pS30-S33, Sept 2011.
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Telemicroscopy Equipment
 Microscope camera (Nikon DS-L2 on Olympus scope)
 Windows IP Configuration
 Controller unit
 Ethernet adaptor
 Local Area Connector
 No special software.
 Consult with vendors.
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Smart Phone Camera Option
 Email digital images to experts for review
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Diagnosing BSI – Accurate GS
 Up to 40% of all patients with blood stream infections
receive inadequate antibiotic treatment until the 1st
notification of a positive blood culture…GS.
 10-20% of patients not started on any antibiotics until GS.
 30-45% require change in empirical treatment.
 Blood GS reported in <1 hour can lead to 17% lower mortality.
 GS accuracy critical for accurate therapy, saves $.
Sogaard, Mette, et al., First Notification of Positive Blood Cultures and High Accuracy of the Gram Stain,
JCM 4-2007, vol 45, no 4, 1113-1117, JCM.
Wolk, Donna, Dunne, Michael W. Jr., New Technologies in Clin Micro, JCM Vol 49, pS62-67, Sept 2011.
Uekahra Yuki, et al, Impact of Reporting GS Results from Blood Culture Bottles on Selection of
Antimicrobial Agents, AJCP, 2009, 132, 18-25.
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Blood Culture GS Diagnosis?
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Diagnosing Bacterial Meningitis
 One of the most important GS in Micro.
 Rapid, accurate ID of the pathogen in 60%–90% of
patients with community-acquired bacterial meningitis,
 97% specificity
- depending on pathogen and prior
treatment.
 Spend extra time searching for pathogen if WBCs
present – can be sparse!
Baron E. et al., IDSA and ASM Guidelines 2013.
Brouwer M.C., et al., Epidemiology, Diagnosis, Antimicrobial Treatment of Acute Bacterial
Meningitis, Clinical Micro Reviews, Vol 23, issue 3, July 2010. www.cdc.gov .
Tunkel, Hartman, Kaplan et al., CID 2004:39, 1 Nov. 1269-70
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CSF Gram Stain Tips
 Cytospin can increase sensitivity 100x !
(more sensitive than bacterial antigen test)
 Acridine orange stain helpful for intracellular
bacteria (i.e. many PMNs and NOS)
 After 1 hour, 32% decrease in WBC detection
 10-20% positive CSF Gram stains have neg
cultures, but blood cultures pos 50-90%.
Chapin-Robertson et al., Cytospin Increases Sensitivity… of CSF, JCM 30:377-380, 1992.
Farin Manian, MD, MPH, Detection and Treatment of CNS Infections, Chief Infect Diseases,
Mercy Hospital, St.Louis,MO April 2012.
Karen Carroll, MD., “Bacterial Meningitis”, ARUP Laboratory, Salt Lake City, March 1996.
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Cytospun Cell Morphology
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CSF Sensitivity
 Gram stain can have up to 93% sensitivity rate,
prior to antibiotic therapy, depending on bacteria:
 Strep pneumo 69-93%
 Strep agalactiae 80-90%
 N.meningitidis 30-89%
 Hemophilus influenzae 25-65%
 Listeria 23-36%
 Staph aureus 20-44%
 Recognize age-related CSF pathogens.
Brouwer M.C., et al., Epidemiology, Diagnosis, Antimicrobial Treatment of Acute Bacterial Meningitis,
Clinical Micro Reviews, Vol 23, issue
3, July 2010.
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Neisseria meningitidis in CSF
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CSF with Strep pneumo
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CSF with E coli
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Cryptococcus in Blood and CSF,
(non-compliant HIV+ patient)
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Diagnosing Fungal Infections –
Are we Searching on Gram Stains?
Harrington, Brian J. et al., Gram Stains – Beyond the Basics, Lab Med, Vol 33, No 8, p609-614, Aug 2002.
Mohan, Subhash, Gram Stain - Looking Beyond Bacteria to Find Fungi in GS Smear, 2009.
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Budding Yeast or Artifact?
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Septate Fungal Elements
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Fatal Fungal Soft-Tissue Infections After a
Tornado - Joplin, Missouri, 2011
 1st known cluster assoc with
tornado victims.
 Lacerations, foreign body, blunt




trauma, fractures.
13 confirmed patients yielded the
Mucormycete Apophysomyces
trapeziformis.
Surgical debridement, culture
positive.
Cutaneous mucormycosis usually
opportunistic in immunocompromised with fatality rate of
29-83%.
MMWR/CDC report:
http://www.faqs.org/periodicals/201107/
2413959571.html#ixzz1Umy2QzZv
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Diagnosing BV
 CAP MIC.22280: detection of Bacterial Vaginosis (BV) by
“Graded Gram stain” for evaluation of vaginal flora.
 Requires pattern recognition, compare ratio of normal flora
(Lactobacillus) to altered flora (Gardnerella, Mobiluncus, other
anaerobes.
 GS is “Gold Standard”, more specific than culture or probe.
 NIH recommends screening in high risk pregnancy.
MMWR CDC Sexually Transmitted Diseases Treatment Guidelines, 2010. www.cdc.gov/std/treatment
IDSA and ASM Guidelines 2013, Baron et al., CID 2013;57.
Hammoud, K.,Treatment of GU Tract Infections: An Evidence Based Approach. 4-13-2012.
Bacterial Vaginosis: An Update on Dx and Rx: Expert Commentary and Five Year Review,
www.MedScape.com
Carol Spiegel, Bacterial Vaginosis, Clinical Micro Review. 1991;4:485-502
Hogan VK et al, Relative performance of 3 methods for diagnosing BV, Maternal Child Health 2007.
Nugent, Krohn, Hillier, Reliability of diagnosing bacterial vaginosis is improved by a standardized method of
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gram stain interpretation. JCM. 1991;29:297-301.
Graded Vaginal Gram Stain
Normal
Abnormal (BV)
CDC MMWR STD Treatment Guidelines
2010,
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Trichomonas on Vaginal Gram Stain
(confirm with acridine orange stain)
ASM Microbe Library
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Gram Stain Case Studies
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Case Study #1
 62 year old with arm infection, called
physician for antibiotic prescription
 Infection spread upwards to shoulder
within several days
 Pt admitted for surgical intervention
 Stat Gram stain of shoulder tissue = Staph
 Surgeon suspects Strep. (No telemicroscopy.)
 Repeat GS = Staph?
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Gram Positive Cocci Pairs
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Case Study #1 (con’t)
 Amputation of arm at shoulder
 Patient expired within 24 hours
 Shoulder tissue and blood cultures grew
Streptococcus pyogenes
 Phage typing = “flesh eating” strain
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Case Study #2
• Healthy 45 year old male pricked thumb on
his metal boot eyelet.
• Within 24 hours, acute thumb pain, low
fever, red streak up arm. Doctor visit.
• Blood cultures drawn, oral antibiotics started.
• Admitted to hospital:
– IV Ampicillin
– surgical debridement of wound,
– culture of drainage
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GPC in Chains in Blood
Note lysed RBCs
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Strep pyogenes
infection, surgical
grafting
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Case Study #3
 Post-menopausal woman with severe headache
and flank pain, low grade fever
 CT scan = walnut size tumor or abscess
 Surgical drainage = purulent material
 Gram stain = short, branching Gram positive rods
 Partial acid-fast stain = negative
 48 hours later, anaerobic culture grew tiny white
molar tooth colonies
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Branching Gram Pos Bacilli
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Case Study #3 (con’t)
 Culture report = Actinomyces israelli and
Fusobacterium
 GS review did not show any fusiform bacteria.
 Source of infection – patient admitted IUD still in
place after 30 years!
 IUD removed and cultured, grew Actino
 Treatment = 3 week course penicillin
 Patient successfully recovered.
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Case Study #4
 44 year old truck driver, severe thigh pain
 History of leukemia, 1 year partial remission
 Examined in ER, dx = muscle cramp.
 Within 24 hrs, returned to ER, X-ray = gas in tissue
= probable cellulitis, gas gangrene
 Surgery sent thigh tissue for stat Gram stain
 GS report = Gram negative rods
 Surgeon questioned result (no telemicroscopy)
 Repeat GS = Gram positive rods?
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Large GPB with Spores in Blood
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Anaerobic Swarming Growth
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Case Study #4 (con’t)
 Leg amputated
 Patient expired < 24 hours post admission
 Thigh tissue and blood cultures grew
Clostridium septicum (anaerobic swarmer)
 Pathogen in patients with hematologic
disorders, endogenous origin.
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Case Study #5
 77 year old diabetic, former cancer patient,
admitted with FUO, altered mental status
 WBC = 9,200 26% bands
 X-rays, ultrasound, CT scan negative
 R/o UTI, pneumonia, meningitis
 Empiric treatment = Rocephin
 Blood culture GS = small gprs and Strep
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Blood with GPCB, GPC in Chains?
Lysed RBCs
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Morphology
Beta hemolysis on blood agar
Intracellular GPB
ASM Microbe Library
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Case Study #5 (con’t)
 Blood cultures grew Listeria monocytogenes
 Listeria can appear as GPCB which can chain up.
 Discussion of recent lunch meat recall prompts patient’s





wife to bring meat samples to lab
Listeria also isolated in pure culture from meat
Health dept notified, USDA collects samples
CDC confirmed different strain, different manufacturer
New official, international recall of meat
Patient recovered despite 25% mortality rate.
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Summary: How can we Meet
Gram Stain Challenges?
• Identify challenges in lab settings
• Bridge gap between core lab and satellites, non-micro techs.
• Partner, communicate, provide support, share expertise.
• Implement quality improvement processes:
• Monitor GS accuracy rate (meet CAP requirements)
• Improve GS expertise with robust training, feedback
•
•
Optimize specimen and stain quality
Target pathogen, clinically relevant results
• Utilize technology – telemicroscopy
• Maximize positive patient outcomes
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Thank you!
Questions?
[email protected]
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